The present invention relates generally to the field of biotechnology and, particularly, to a method for inhibiting the binding activity of immunoglobulins with immunogens, a method for inhibiting culture agglutination and the actual inhibitors.
Many forms of immune proteins exist in mammals including humans, and other classes of animals having immune systems. It is generally believed that the immune proteins IgM and IgG are the most important proteins that provide protection against microbial invasion of mammals. IgM is similar to IgG but is composed of five IgG-like molecules in a pentamer arrangement. The immunoglobulins IgM and IgG are circulated in the blood to combat invasion by foreign matter, mainly bacteria and are transferred to milk to establish immunity in nursing offspring.
Scientific literature suggests that the immune proteins IgM and IgG interact when a binding site on the variable regions of the light and heavy chains (see FIG. 1) attaches to an antigenic site or to a complimentary binding site on the tail of the heavy chain (Fc fraction) of another IgM or IgG molecule. Many researchers believe that IgM and IgG lose their interactive ability when the heavy chains are removed from the Fab fractions. Our work in the development of the present invention, however, indicates that the carbohydrate moieties or constant regions on both the light and heavy chains of immune proteins are capable of interacting with antigenic sites and/or bridging compounds thereby causing immune reactions and agglutination. Thus, it should be appreciated, that simple cleavage of the Fab fraction is not effective to inhibit immune reactions. Accordingly, a new method must be developed to achieve this end.
Accordingly, it is a primary object of the present invention to provide a method for inhibiting the binding activity of immunoglobulins with immunogens. Such a method has a number of beneficial applications including, but not limited to, inhibiting culture agglutination in a fermentation medium and blocking selected antigenic sites so as to prevent problematic immune reactions in the health care field.
Yet another object of the present invention is to provide a method for inhibiting culture agglutination in a fermentation medium wherein agglutination of the microorganisms responsible for fermentation is substantially prevented. Such a method allows, for example, more efficient production of a more consistent and higher quality fermentation product (e.g. cheddar cheese, cottage cheese).
Still another object of the present invention is to provide a method for inhibiting the binding activity of mammalian immunoglobulins and in particular IgA, IgG and IgM in a biologically safe and effective manner. Such an approach may be utilized to limit or prevent immune reactions and prevent infection and, accordingly, are beneficial for a number of uses in the care and treatment of patients suffering from a number maladies.
Yet another object of the present invention is to provide inhibitors of the binding activity of immunoglobulins including mammalian immunoglobulins and particularly IgA, IgG and IgM. This is accomplished through the utilization of corresponding immunoglobulins hydrolyzed with plant proteases to provide peptides having a molecular weight of less than 10,000 Daltons and preferably of about 1,000 Daltons.
Additional objects, advantages and other novel features of the invention will be set forth in part in the description that follows and in part will become apparent to those skilled in the art upon examination of the following or may be learned with the practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention as described herein, an improved method is provided for inhibiting the binding activity of immunoglobulins with immunogens. The method is particularly adapted for utilization in inhibiting the binding activity of mammalian immunogens including, specifically, IgA, IgG and IgM immune proteins. The method broadly includes the initial step of treating a source of immunoglobulins with an enzyme that hydrolizes the immunoglobulins into immunoreactive peptides having a molecular weight of less than 10,000 Daltons. Preferably, the enzyme is a plant protease that cleaves the immunoglobulins, IgA, IgG and IgM, above the second disulfide bond (see FIG. 1). This results in the liberation of two Fab fractions. The enzymes papain, ficin and bromelain are particularly useful for this purpose.
In addition to cleaving the immunogens IgA, IgG and IgM above the second disulfide bond, the hydrolysis is continued for an additional period of time to clip and remove the carbohydrate moiety or constant regions from the variable regions so as to provide a peptide fraction having a molecular weight of less than 10,000 Daltons. This may be accomplished through ultrafiltering the source of immunoglobulins as they are hydrolized with the enzyme. A diafiltration technique may be utilized so that the smaller peptide fractions produced as a result of hydrolysis (i.e. those under 10,000 Daltons) pass as permeate through a polysulfone or other membrane having a molecular weight cut off of 10,000 Daltons. Specifically, to achieve this end, hydrolysis preferably is completed at a temperature between 30-50xc2x0 C. for a period of at least two hours. As permeate is removed from the source of immunoglobulins undergoing hydrolysis a buffer of equivalent volume may be added.
The permeate, consisting of peptides of a molecular weight of less than 10,000 Daltons and preferably approximately 1,000 Daltons is then collected. Following collecting is the reacting of immunogens with the peptides so as to bind and block antigenic sites on the immunogens. Then follows the introducing of the peptide bound and blocked immunogens into the mammal. Advantageously, the bound and blocked antigenic sites on the immunogens make the immunogens substantially xe2x80x9cinvisiblexe2x80x9d to the immune system of the mammal thereby preventing undesired and problematic immune reactions from occurring.
Such an approach has a number of applications including, specific use in the artificial insemination of mammals including humans and other animals. Specifically, the sperm cells to be inseminated are reacted with the peptide so as to bind and block the antigenic sites or determinants on the sperm cells. When subsequently inseminated into a recipient, the immune reaction of the recipient is reduced and/or substantially eliminated, and accordingly, the aggregation of sperm cells is virtually prevented. Thus, the sperm cells remain free to reach the ovum for purposes of fertilization.
In accordance with yet another aspect of the present invention, a method for inhibiting culture agglutination in a fermentation medium including immunogens is provided. As before, this method includes an initial step of treating a source of immunoglobulins with an enzyme such as a plant protease to hydrolize the immunoglobulins into peptides having a molecular weight of less than 10,000 Daltons and preferably, approximately 1,000 Daltons. Specifically, as previously described these peptides are produced utilizing ultrafiltration and diafiltration techniques. Preferably, papain, ficin and/or bromelain is used as the enzyme.
The immunoglobulins may be obtained from a number of sources including, but not limited to, raw whey, dry whey, whey protein isolates, non-fat-dried milk, blood serum protein isolates, purified immunogen preparations and mixtures thereof.
Following hydrolysis is the collecting of the peptides produced as a result of the treatment of the immunoglobulins with papain, ficin and/or bromelain and in accordance with the ultrafiltration and diafiltration techniques. Specifically, the resulting peptides present in the permeate may be concentrated utilizing reverse osmosis or evaporation and then dried using freeze drying, spray drying or vacuum drying techniques. These dried preparations can then be formulated into existing culture media preparations.
The peptides in these culture media preparations are then utilized by inoculating a growth media for microorganisms, useful in fermenting the fermentation medium, with the peptides. Next is the growing of the microorganisms in the inoculated growth media whereby the peptides bind antigenic sites on the microorganisms. This is then followed by adding the microorganisms grown in the inoculated growth media to the fermentation medium whereby the peptide bound antigenic sites on the microorganisms block the immunoglobulins in the fermentation medium from binding those antigenic sites and thereby causing agglutination of the microorganisms.
Such a method is particularly useful in the production of cottage, cheddar and other cheeses wherein agglutination is a significant problem leading to revenue loss. Specifically, agglutination results in uneven distribution of starter culture throughout the milk and uneven acid production in the cheese vat. Grainy, shattered curd, sediment formation and slow acid production are all problems that directly relate to agglutination of lactic acid starter strains. Accordingly, direct consequences of agglutination are inconsistent product quality and yield losses which both lead to decreased profits. Accordingly, as the present invention addresses and largely relieves the agglutination problem, it represents a significant advance in the art.
In accordance with still another aspect of the present invention, an inhibitor is provided for inhibiting the binding activity of immunoglobulins and particularly mammalian immunoglobulins including IgA, IgG and IgM. Specifically, the inhibitor is derived from a source of immunoglobulins hydrolized with a plant protease such as papain, ficin and/or bromelain and then ultrafiltered with or without diafiltering to provide peptides having a molecular weight of less than 10,000 Daltons and preferably approximately 1,000 Daltons. Such an inhibitor will have a number of unique biotechnological applications such as in the health care field where it is particularly desirable to block problematic immune reactions.
Still other objects of the present invention will become apparent to those skilled in this art from the following description wherein there is shown and described a preferred embodiment of this invention, simply by way of illustration of one of the modes best suited to carry out the invention. As it will be realized, the invention is capable of other different embodiments and its several details are capable of modification in various, obvious aspects all without departing from the invention. Accordingly, the drawings and descriptions will be regarded as illustrative in nature and not as restrictive.